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Callum O'Dowda and Kemar Roofe have been sold in the space of a week for what could rise to a combined £4.6m.
And Appleton says finding a replacement for Republic of Ireland midfielder O'Dowda is now his immediate priority.
"We've lost the best part of 40 goals in the last week, so we need some more additions," he told BBC Radio Oxford.
O'Dowda, 21, joined Championship Bristol City on Thursday in a three-year deal while Roofe moved to Leeds United last week for £3m.
"Callum has ambitions to play in the Championship," Appleton said. "He made that pretty clear.
"The irony is I've had conversations with a number of players over the last couple of months on a similar subject and I believe we as a club can match those ambitions.
"That's the frustration from my point of view. I was going to build a team this season around Callum."
O'Dowda scored 10 goals for Oxford United last season during their automatic promotion from League Two and run to the Football League Trophy final.
Appleton, who has already brought in eight players this summer, has plans in place to further bolster the squad.
Among his targets had been Bristol Rovers striker Matt Taylor, before the 26-year-old agreed a new deal with Rovers on Friday.
"It's an interesting one as without speaking too much out of turn, I did think we'd shook hands on an agreement," he said.
"But I'm being told by his representative that he hasn't got a desire to play for his hometown club.
"They'll be players out there who will become available," Appleton added. "That might not be until later in the window or it could be as quick as the next 24 hours."
Oxford United manager Michael Appleton says they cannot afford to let any more players leave the club if they are to be competitive in League One.